L14 - EPEC & enteric disease Flashcards
What is EPEC?
Enteropathogenic E. coli (EPEC)
Major cause of watery diarrhoea in infants (mostly <6-month-old)
Its primarily now a problem for developing countries – main issue due to hygiene
What is EPEC a member of?
These bacteria are a member of the attachment & effacing family of pathogens – they cause changes on the cells, the attaching & facement of the absorbance to microvilli
How does EPEC infect host cells?
- The bacteria target the absorbent epithelia of the small intestine
- Most bacteria cannot get through this – however EPEC can sink into the brush border of microvilli
- Make contact with the host plasma membrane
- This triggers signaling responses in the host cell that lead to the formation of pedestal-like structures
- Also associated with the elongation of the microvilli and the loss of microvilli which is thought to allow more bacteria to be recruited to the site of infection
What is virulence of EPEC mainly dependent on?
The key determinant is a secretory system – the ability to be able to transfer proteins outside or into host cells is important in the disease process
They do this by a T3SS (type 3 Secretion system) – which is a molecular needle syringe like structure which connects the cytoplasm of the host and bacteria
A novelty of the EPEC is that the needle is extended by one of the secretory proteins (EspA) polymerizing to make a long extension
Where is T3SS encoded?
On the Locus of Enterocyte Effacement (LEE) region
LEE is a pathogenicity island
Structure of T3SS
LEE has about 40 proteins - half of them form the T3SS
Some of the proteins are secreted – secreted translocator proteins – they form the extension of the T3SS – allows the system to interact with the host plasma membrane – inserts 2 proteins into the host plasma membrane to generate a pore – allows effector proteins to be delivered into the host cell – these drive the disease process
Known that the protein in the plasma membrane (EspB) also has effector functions
There are other proteins involved in the system – regulators & chaperones
Intimin is an outer membrane protein that is critical for disease
How did EPEC become pathogenic?
EPEC pathogenesis driven by acquisition of new DNA
How can you treat EPEC diseases?
Can treat EPEC diseases quite easily
BUT
Rapid acquisition of antibiotic resistance by the transferring of plasmids
What are Non-LEE-encoded (Nle) effectors?
While some secreted effector proteins are encoded on a pathogenicity island known as LEE, several effector molecules are encoded outside the LEE (Nle)
Has been found that a subset of these have other effector molecules that are delivered by the T3SS
They have 17 Nle effector molecules
Bacteria can deliver 24 effector molecules into the host cell
Gives it a huge capacity to alter host cellular processes
In vitro Model of EPEC’s Target Cell: Absorptive epithelial (Enterocytes)
Important that studies are done of the cell types that the bacteria actually affect – need the key physiology of the target cell
Caco2 model has an insert which has a placid membrane with little holes in – not big enough for cells to fall out but it allows them to feed from both sides
How do you get Caco 2 model cells?
Can take these cells from a cancer – been found you can grow them for 2/3 weeks and they spontaneously differentiate into cells that morphologically & physiologically mimics EPECs in vivo target
How does the Caco 2 model work?
- Can add the bacteria to the top compartment (apical chamber) – equivalent to the lumen of the gastrointestinal tract & the bottom is equivalent to the blood system
- Can scan the surface of the cells – can see bacteria bound to the membrane
- Can see the elongation of the microvilli
- Areas where there’s no microvilli as its been removed (faced)
- In later timepoints there is lots of bacteria colonies & no microvilli
How can you use a voltmeter in the Caco 2 model?
Can also use a voltmeter to look at the ability of current to travel to one compartment to the next – reflects the tightness of the cells
- If the cells are tight together the current is very slow
- If they’re disrupted the current moves
- When you effect EPEC you get rapid loss of electrical resistance
- Bacteria can disrupt these tight junctions
Diarrhoeal-associated alteration of enterocytes
LEE-encoded effector activity linked to diarrhoeal-associated alterations
Can be done by 3 mechanisms:
• Alter activity of transporters in the microvilli
• Remove absorbed microvilli
• Disrupt tight junctions between cells
LEE-encoded effector activity linked to diarrhoeal-associated alterations
Mechanism:
Alter activity of transporters in the microvilli
Cause them to export more ions or inhibit their ability to take up ions – to do with water balance
Bacteria alter the transporter in the apical membrane to explain the rapid onset of diarrhea
A/E lesion formation is linked to loss of major water transporter from absorptive microvilli
LEE-encoded effector activity linked to diarrhoeal-associated alterations
Mechanism:
Remove absorbed microvilli
This reduces SA of the GI tract & remove the transporters – promotes diarrhoea & malnutrition
LEE-encoded effector activity linked to diarrhoeal-associated alterations
Mechanism:
Disrupt tight junctions between cells
Allows the unregulated movement of ions & antigens between the cells – if they can move in an unregulated manner then water can follow it and promote diarrhoeal disease
What does EPEC disease depend on?
Intimin & Tir (transmembrane intimin receptor) are critical for the disease process
Bacteria understand the host physiology & have exploited it by stealing concepts – bacteria delivers its own receptor
How have bacteria stolen the receptor-ligand concept from Intimin-Tir association?
The Tir effector molecules expressed in the bacterial cytoplasm & transferred via the T3SS into the host cytoplasm where it recognises the host kinases which modify specific residues – associated with Tir being inserted into the host plasma membrane in this hairpin like structure
The middle bit is a binding site for the surface protein intimin – once inserted it is recognised by host tyrosine kinases which phosphorylate specific tyrosine residues
Following interaction, it causes clustering of the Tir receptor leading to phosphorylation of the tyrosine’s causing a signalling cascade – recruits host protein called Nik (adaptor molecule) whose function it to recruit N-WASP which activates the actin nucleating machinery called Arp2/3 which leads to these pedestal-like structures underneath the bacteria
This demonstrates how the bacteria understands the host cell physiology
• Exploit host kinases to trigger its signalling cascades to promotes its life cycle
How is EPEC disease dependent on Tir-Intimin (receptor-ligand) interaction
A lot of Tirs ability to alter host cellular processes depends on it binding intimin – triggers a signaling cascade
Bacteria from outside the cell, can keep control of its effector molecules through the Tir-Intimin interaction
They allow the bacteria to sink through the absorbed microvilli – if they can’t do this they can’t deliver their effector molecules in the cytoplasm
What is microvilli size dependent on?
Threadmilling
Where actin is added & polymerized at the tip, the rate of adding vs the rate of natural turnover at the other end is threadmilling – can be altered by promoting removal or addition of actin
What proteins do many pathogens target?
Small RhoGTPases are a key target of many pathogens as they play a central role in controlling different signaling cascades that the bacteria manipulate
What is MAP?
Map is a Cdc42 specific GEF – molecular ON/OFF switches
Cdc42 is a member of the small RhoGTPase family of molecular on-off switches of signaling cascades
When receptor signaling occurs, triggers signaling cascade that activates small GTPase via GEF and then proteins like GAF switch them off again
What is EspF?
EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC)
How do EspF & MAP share a common target?
- Structural studies have shown that Map is GEF – it functions specifically to activate Cdc42 which triggers the signaling cascade leading to these filopodia formation
- It’s also been found that EspF directly activates N-WASP as it has 3 poly proline rich regions – each one has an N-WASP binding domain – this recruits N-WASP
- Found that the feature is very identical to the auto-inhibitory domain in N-WASP, it binds & opens N-WASP to activate it – makes it constitutionally active to drive actin nucleation events
- Also found that N-WASP has recruitment sites for a protein called SNX9 which is linked the recruitment of the endocytic machinery to internalize the membrane
- Illistrates the knowledge of the bacteria
Rapid onset of watery bacteria
Tir, Intimin, Map and EspF cooperativity removes major water pump from intact microvilli
The rapid onset of watery diarrhoea is related to the SGLT1
How does EPEC remove SGLT1?
- When you infect with EPEC there is a rapid loss of SGLT1 from the apical surface
- Effector molecules work together to remove SGLT1 from the surface
- We think EspF is recruiting SNX9 in N-WASP to drive endocytosis - & the other effector molecules must be part of this process
- We know that the EspG LEE effector can prevents secretory pathways – prevents new SGLT1s being added to the microvilli
- This causes the rapid onset of watery diarrhea
Paradox in EPEC human disease
- EPEC disrupts cell-cell junctions in vitro and in vivo models – this allows antigens such as flagellin (key stimuluses of inflammation) to get between the cells & interact with its TLR, TLR5
- This then triggers signalling cascades that leads to the activation of transcription factors to trigger the expression of cytokines such as IL-8 which act as alarm calls to recruit PMNs to the site of infection so they can start attacking the bacteria
- These should lead to the release of more chemokines which leads to an inflammatory diarhhoeal disease
- EPEC diseases are not associated with a strong inflammatory response which suggests there is a suppressive mechanism
Mechanisms employed by EPEC to inhibit infection-induced gene transcriptional responses
NleC was found to interact with NF-kB in the nucleus and was able to cleave it as it is a metalloprotease
The complex was recruited by docking at the NleC and then has a catalytic domain that cleaves NF-kB to stop it being functional
Loss of IκB and NF-κB Rel proteins p65 and p50
When they looked at the crystal structure of NleC, it mimics the target of NF-kB
Again illustrates how bugs understand the physiology & can mimic the DNA
EPEC Interferes with the activity of the NF-кB cofactor RPS3 by multiple mechanisms
- RPS3 directs NF-kB to specific genes
- Its been found that to the effector molecules, that the pathogens look like serine-threonine kinases – have stolen the structure & put a signal sequence on it & delivered it back into the host to utilize it against the host
- Found that the molecule interferes with the function of ribosome binding protein S3 although doesn’t require kinase activity
- Also found that NleH could interfere with the function of RPS3
- Illustrates that bacteria have the ability to alter specific proteins that hinder the replication pathway
EPEC Deploys the protease strategy to inhibit Map Kinase Signalling Pathways
They can target other effector molecules that cleave other molecules
NleCs will go and cleave JNK & p38 – because they recognize the loop structure – a residue difference in ERK protects ERK from being degraded
However, ERK is stopped by other effector molecules
Other Targets on the Receptor-IKK-NFкB activation pathway: NleE
Another target molecule targets the gene upstream from that – TAB proteins
They are important for activating the kinase
NleE catalyses the cysteine methylation of these 2 TAB proteins – prevents capacity of proteins to recruit TAK1
Other Targets on the Receptor-IKK-NFкB activation pathway: Tir
Another effector molecule that targets the upstream protein TRAF6
Tir can target these proteins & lead to their degradation and other TRAFs which are associated with cytokine signaling
Tir also has other functions to inhibit the pathway – some residues on the C-terminus that are phosphorylating by the host & act as an ITIM, which recruits phosphatase to inhibit TRAF function so it doesn’t get ubiquitinated so cannot recruit the TABs – inhibits NF-kB signaling
Other Targets on the Receptor-IKK-NFкB activation pathway: NleB
NleB targets proteins quite high in the pathway
Can inhibit signaling though receptor kinases but not through antigens like the TLRs suggesting its targeting something upstream of these pathways
NleB targets a specific component upstream, causing the modification of these domains – TRA domains
Adds a sugar onto arginine (novel modification) on the TRA domain which inhibits its activity
The target arginine is conserved in multiple proteins involved in regulating cell death pathways
Other Targets on the Receptor-IKK-NFкB activation pathway: EspL
EspL is a cysteine protease effector molecule that targets proteins with the rim motif
Found in lots of different molecules & important in signaling through NF-kB & also protects the cell death signaling pathways
How do bacteria survive without killing the host?
if you over inhibit one pathway, the cell recognizes that and causes dysfunction leading to cell death
• The bacteria understand this
• They target lots of proteins at low levels to inhibit their activity a little bit – doesn’t impact any individual step but collectively they inhibit signaling to NF-kB
• The levels of delivery are critical
• Also gives the bacteria evolutionary advantages
No pathogen wants to kill its host, it just wants to have a maximal level where it can propagate before its killed by the cell
Can lead to oversignalling & death if you’re immunocompromised or if the pathogen is extra virulent
